Prevention and treatment of pain using antibodies to sphingosine-1-phosphate

ABSTRACT

The present invention relates to use of anti-S1P agents, for example, humanized monoclonal antibodies, for prevention and/or treatment of pain, including neuropathic pain, hyperalgesia, allodynia, and chemotherapy-induced pain.

RELATED APPLICATIONS

This application is a continuation of, and claims the benefit of andpriority to, U.S. non-provisional patent application Ser. Nos.13/246,704 filed 27 Sep. 2011 (attorney docket no. LPT-3010-CP3), and12/258,383, filed on 24 Oct. 2008 and issued on 27 Sep. 2011 as U.S.Pat. No. 8,026,342, (attorney docket no. LPT-3010-CP), the contents ofeach of which are herein incorporated by reference in its entirety, forany and all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to agents that bindsphingosine-1-phosphate (S1P), particularly to humanized monoclonalantibodies, antibody fragments, and antibody derivatives specificallyreactive to S1P under physiological conditions. Such agents can be usedin the treatment and/or prevention of various diseases or conditionsthrough the delivery of pharmaceutical compositions that contain suchagents.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein, or any publication specifically orimplicitly referenced herein, is prior art, or even particularlyrelevant, to the presently claimed invention.

2. Background

Bioactive Signaling Lipids

Lipids and their derivatives are now recognized as important targets formedical research, not as just simple structural elements in cellmembranes or as a source of energy for β-oxidation, glycolysis or othermetabolic processes. In particular, certain bioactive lipids function assignaling mediators important in animal and human disease. Although mostof the lipids of the plasma membrane play an exclusively structuralrole, a small proportion of them are involved in relaying extracellularstimuli into cells. “Lipid signaling” refers to any of a number ofcellular signal transduction pathways that use cell membrane lipids assecond messengers, as well as referring to direct interaction of a lipidsignaling molecule with its own specific receptor. Lipid signalingpathways are activated by a variety of extracellular stimuli, rangingfrom growth factors to inflammatory cytokines, and regulate cell fatedecisions such as apoptosis, differentiation and proliferation. Researchinto bioactive lipid signaling is an area of intense scientificinvestigation as more and more bioactive lipids are identified and theiractions characterized.

Examples of bioactive lipids include the eicosanoids (including thecannabinoids, leukotrienes, prostaglandins, lipoxins,epoxyeicosatrienoic acids, and isoeicosanoids), non-eicosanoidcannabinoid mediators, phospholipids and their derivatives such asphosphatidic acid (PA) and phosphatidylglycerol (PG), plateletactivating factor (PAF) and cardiolipins as well as lysophospholipidssuch as lysophosphatidyl choline (LPC) and various lysophosphatidicacids (LPA). Bioactive signaling lipid mediators also include thesphingolipids such as sphingomyelin, ceramide, ceramide-1-phosphate,sphingosine, sphingosylphosphoryl choline, sphinganine,sphinganine-1-phosphate (Dihydro-S1P) and sphingosine-1-phosphate.Sphingolipids and their derivatives represent a group of extracellularand intracellular signaling molecules with pleiotropic effects onimportant cellular processes. Other examples of bioactive signalinglipids include phosphatidylserine (PS), phosphatidylinositol (PI),phosphatidylethanolamine (PEA), diacylglyceride (DG), sulfatides,gangliosides, and cerebrosides.

Sphingolipids are a unique class of lipids that were named, due to theirinitially mysterious nature, after the Sphinx Sphingolipids wereinitially characterized as primary structural components of cellmembranes, but recent studies indicate that sphingolipids also serve ascellular signaling and regulatory molecules (Hannun, et al., Adv. LipidRes. 25:27-41, 1993; Speigel, et al., FASEB J. 10:1388-1397, 1996;Igarashi, J. Biochem 122:1080-1087, 1997; Hla, T. (2004). Semin Cell DevBiol, 15, 513-2; Gardell, S. E., Dubin, A. E. & Chun, J. (2006). TrendsMol Med, 12, 65-75). Sphingolipids are primary structural components ofcell membranes that also serve as cellular signaling and regulatorymolecules (Hannun and Bell, Adv. Lipid Res. 25: 27-41, 1993; Igarashi,J. Biochem 122: 1080-1087, 1997). The sphingolipid signaling mediators,ceramide (CER), sphingosine (SPH) and sphingosine-1-phosphate (S1P),have been most widely studied and have recently been appreciated fortheir roles in the cardiovascular system, angiogenesis and tumor biology(Claus, et al., Curr Drug Targets 1: 185-205, 2000; Levade, et al.,Circ. Res. 89: 957-968, 2001; Wang, et al., J. Biol. Chem. 274:35343-50, 1999; Wascholowski and Giannis, Drug News Perspect. 14:581-90, 2001; Spiegel, S. & Milstien, S. (2003).Sphingosine-1-phosphate: an enigmatic signaling lipid. Nat Rev Mol CellBiol, 4, 397-407).

For a review of sphingolipid metabolism, see Liu, et al., Crit Rev.Clin. Lab. Sci. 36:511-573, 1999. For reviews of the sphingomyelinsignaling pathway, see Hannun, et al., Adv. Lipid Res. 25:27-41, 1993;Liu, et al., Crit. Rev. Clin. Lab. Sci. 36:511-573, 1999; Igarashi, J.Biochem. 122:1080-1087, 1997; Oral, et al., J. Biol. Chem.272:4836-4842, 1997; and Spiegel et al., Biochemistry (Moscow) 63:69-83,1998.

S1P is a mediator of cell proliferation and protects from apoptosisthrough the activation of survival pathways (Maceyka, et al. (2002),BBA, vol. 1585): 192-201, and Spiegel, et al. (2003), Nature ReviewsMolecular Cell Biology, vol. 4: 397-407). It has been proposed that thebalance between CER/SPH levels and S1P provides a rheostat mechanismthat decides whether a cell is directed into the death pathway or isprotected from apoptosis. The key regulatory enzyme of the rheostatmechanism is sphingosine kinase (SPHK) whose role is to convert thedeath-promoting bioactive signaling lipids (CER/SPH) into thegrowth-promoting S1P. S1P has two fates: S1P can be degraded by S1Plyase, an enzyme that cleaves S1P to phosphoethanolamine andhexadecanal, or, less common, hydrolyzed by S1P phosphatase to SPH.

The pleiotropic biological activities of S1P are mediated via a familyof G protein-coupled receptors (GPCRs) originally known as EndothelialDifferentiation Genes (EDG). Five GPCRs have been identified ashigh-affinity S1P receptors (S1PRs): S1P₁/EDG-1, S1P₂/EDG-5, S1P₃/EDG-3,S1P₄/EDG-6, and S1P₅/EDG-8 only identified as late as 1998 (Lee, et al.,1998). Many responses evoked by S1P are coupled to differentheterotrimeric G proteins (G_(q-, G) _(i), G₁₂₋₁₃) and the small GTPasesof the Rho family (Gardell, et al., 2006).

In the adult, S1P is released from platelets (Murata et al., 2000) andmast cells to create a local pulse of free S1P (sufficient enough toexceed the K_(d) of the S1PRs) for promoting wound healing andparticipating in the inflammatory response. Under normal conditions, thetotal S1P in the plasma is quite high (300-500 nM); however, it has beenhypothesized that most of the S1P may be ‘buffered’ by serum proteins,particularly lipoproteins (e.g., HDL>LDL>VLDL) and albumin, so that thebio-available S1P (or the free fraction of S1P) is not sufficient toappreciably activate S1PRs (Murata et al., 2000). If this were not thecase, inappropriate angiogenesis and inflammation would result.Intracellular actions of S1P have also been suggested (see, e.g.,Spiegel S, Kolesnick R (2002), Leukemia, vol. 16: 1596-602; Suomalainen,et al (2005), Am J Pathol, vol. 166: 773-81).

Widespread expression of the cell surface S1P receptors allows S1P toinfluence a diverse spectrum of cellular responses, includingproliferation, adhesion, contraction, motility, morphogenesis,differentiation, and survival. This spectrum of response appears todepend upon the overlapping or distinct expression patterns of the S1Preceptors within the cell and tissue systems. In addition, crosstalkbetween S1P and growth factor signaling pathways, includingplatelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF), and basic fibroblastic growth factor (bFGF), haverecently been demonstrated (see, e.g., Baudhuin, et al. (2004), FASEB J,vol. 18: 341-3). The regulation of various cellular processes involvingS1P has particular impact on neuronal signaling, vascular tone, woundhealing, immune cell trafficking, reproduction, and cardiovascularfunction, among others. Alterations of endogenous levels of S1P withinthese systems can have detrimental effects, eliciting severalpathophysiological conditions, including cancer, inflammation,angiogenesis, heart disease, asthma, and autoimmune diseases.

A recent novel approach to the treatment of various diseases anddisorders, including cardiovascular diseases, cerebrovascular diseases,and various cancers, involves reducing levels of biologically availableS1P, either alone or in combination with other treatments. Whilesphingolipid-based treatment strategies that target key enzymes of thesphingolipid metabolic pathway, such as SPHK, have been proposed,interference with the lipid mediator S1P itself has not until recentlybeen emphasized, largely because of difficulties in directly mitigatingthis lipid target, in particular because of the difficulty first inraising and then in detecting antibodies against the S1P target.

Recently, the generation of antibodies specific for S1P has beendescribed. See, e.g., commonly owned, U.S. patent application Serial No.20070148168; WO2007/053447. Such antibodies, which can, for example,selectively adsorb S1P from serum, act as molecular sponges toneutralize extracellular S1P. See also commonly owned U.S. Pat. Nos.6,881,546 and 6,858,383 and U.S. patent application Ser. No. 10/029,372.SPHINGOMAB™, the murine monoclonal antibody (mAb) developed by Lpath,Inc. and described in certain patents or patent applications listedabove, has been shown to be effective in models of human disease. Insome situations, a humanized antibody may be preferable to a murineantibody, particularly for therapeutic uses in humans, wherehuman-anti-mouse antibody (HAMA) response may occur. Such a response mayreduce the effectiveness of the antibody by neutralizing the bindingactivity and/or by rapidly clearing the antibody from circulation in thebody. The HAMA response can also cause toxicities with subsequentadministrations of mouse antibodies.

A humanized anti-S1P antibody has been developed by Lpath, Inc. Thisantibody has all the advantages of the murine mAb in terms of efficacyin binding S1P, neutralizing S1P and modulating disease states relatedto S1P, but with none of the potential disadvantages of the murine mAbwhen used in a human context. As described in the examples hereinbelow,this humanized antibody (referred to as LT1009 or sonepcizumab) has infact shown activity greater than that of the parent (murine) antibody inanimal models of disease.

Definitions

Before describing the instant invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification, asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings In theevent of conflict, the present specification, including definitions,will control.

An “immune-derived moiety” includes any antibody (Ab) or immunoglobulin(Ig), and refers to any form of a peptide, polypeptide derived from,modeled after or encoded by, an immunoglobulin gene, or a fragment ofsuch peptide or polypeptide that is capable of binding an antigen orepitope (see, e.g., Immunobiology, 5th Edition, Janeway, Travers,Walport, Shlomchiked. (editors), Garland Publishing (2001)). In thepresent invention, the antigen is a bioactive lipid molecule.

An “anti-S1P antibody” or an “immune-derived moiety reactive againstS1P” refers to any antibody or antibody-derived molecule that binds S1P.As will be understood from these definitions, antibodies orimmune-derived moieties may be polyclonal or monoclonal and may begenerated through a variety of means, and/or may be isolated from ananimal, including a human subject.

A “bioactive lipid” refers to a lipid signaling molecule. In general, abioactive lipid does not reside in a biological membrane when it exertsits signaling effects, which is to say that while such a lipid speciesmay exist at some point in a biological membrane (for example, a cellmembrane, a membrane of a cell organelle, etc.), when associated with abiological membrane it is not a “bioactive lipid” but is instead a“structural lipid” molecule. Bioactive lipids are distinguished fromstructural lipids (e.g., membrane-bound phospholipids) in that theymediate extracellular and/or intracellular signaling and thus areinvolved in controlling the function of many types of cells bymodulating differentiation, migration, proliferation, secretion,survival, and other processes. In vivo, bioactive lipids can be found inextracellular fluids, where they can be complexed with other molecules,for example serum proteins such as albumin and lipoproteins, or in“free” form, i.e., not complexed with another molecule species. Asextracellular mediators, some bioactive lipids alter cell signaling byactivating membrane-bound ion channels or G-protein coupled receptorsthat, in turn, activate complex signaling systems that result in changesin cell function or survival. As intracellular mediators, bioactivelipids can exert their actions by directly interacting withintracellular components such as enzymes and ion channels.Representative examples of bioactive lipids include LPA and S1P.

The term “therapeutic agent” means an agent to mitigate angiogenesisand/or neovascularization, e.g., CNV and BV maturation, edema, vascularpermeability and fibrosis, fibrogenesis and scarring associated with, orpart of the underlying pathology of, ocular diseases and conditions.

The term “combination therapy” refers to a therapeutic regimen thatinvolves the provision of at least two distinct therapies to achieve anindicated therapeutic effect. For example, a combination therapy mayinvolve the administration of two or more chemically distinct activeingredients, for example, an anti-LPA antibody and an anti-S1P antibody.Alternatively, a combination therapy may involve the administration ofan immune-derived moiety reactive against a bioactive lipid and theadministration of one or more other medicaments, such as pain reliefagents. Combination therapy may, alternatively, involve administrationof an anti-lipid antibody together with the delivery of anothertreatment, such as radiation therapy and/or surgery. Further, acombination therapy may involve administration of an anti-lipid antibodytogether with one or more other biological agents (e.g., anti-VEGF,TGFβ, PDGF, or bFGF agent), other medicaments and another treatment suchas radiation and/or surgery. In the context of combination therapy usingtwo or more chemically distinct active ingredients, it is understoodthat the active ingredients may be administered as part of the samecomposition or as different compositions. When administered as separatecompositions, the compositions comprising the different activeingredients may be administered at the same or different times, by thesame or different routes, using the same of different dosing regimens,all as the particular context requires and as determined by theattending physician. Similarly, when one or more anti-lipid antibodyspecies, for example, an anti-S1P antibody, alone or in conjunction withone or more medicaments are combined with, for example, radiation and/orsurgery, the drug(s) may be delivered before or after surgery orradiation treatment.

An “anti-S1P agent” refers to any agent that is specifically reactive toS1P, and includes antibodies or antibody-derived molecules ornon-antibody-derived moieties that bind S1P and its variants.

A “hapten” refers to a molecule adapted for conjugation to a hapten,thereby rendering the hapten immunogenic. A representative, non-limitingclass of hapten molecules is proteins, examples of which includealbumin, keyhole limpet hemocyanin, hemaglutanin, tetanus, anddiphtheria toxoid. Other classes and examples of hapten moleculessuitable for use in accordance with the invention are known in the art.These, as well as later discovered or invented naturally occurring orsynthetic haptens, can be adapted for application in accordance with theinvention.

The term “chemotherapeutic agent” means anti-cancer and otheranti-hyperproliferative agents. Put simply, a “chemotherapeutic agent”refers to a chemical intended to destroy cells and tissues. Such agentsinclude, but are not limited to: (1) DNA damaging agents and agents thatinhibit DNA synthesis: e.g., anthracyclines (e.g., doxorubicin,donorubicin, epirubicin), alkylating agents (e.g., bendamustine,busulfan, carboplatin, carmustine, cisplatin, chlorambucil,cyclophosphamide, dacarbazine, hexamethylmelamine, ifosphamide,lomustine, mechlorethamine, melphalan, mitotane, mytomycin, pipobroman,procarbazine, streptozocin, thiotepa, and triethylenemelamine), platinumderivatives (e.g., cisplatin, carboplatin, cisdiamminedichloroplatinum), telomerase and topoisomerase inhibitors(e.g., Camptosar), (2) tubulin-depolymerizing agents: e.g., taxoids(e.g., Paclitaxel, docetaxel, BAY 59-8862), (3) anti-metabolites such ascapecitabine, chlorodeoxyadenosine, cytarabine (and its activated form,ara-CMP), cytosine arabinoside, dacabazine, floxuridine, fludarabine,5-fluorouracil, 5-DFUR, gemcitibine, hydroxyurea, 6-mercaptopurine,methotrexate, pentostatin, trimetrexate, and 6-thioguanine (4)anti-angiogenics (e.g., Avastin, thalidomide, revlimid, sunitinib,lenalidomide), vascular disrupting agents (e.g., flavonoids/flavones,DMXAA, combretastatin derivatives such as CA4DP, ZD6126, AVE8062A,etc.), (5) biologics such as antibodies or antibody fragments (e.g.,Herceptin, Avastin, Panorex, Rituxan, Zevalin, Mylotarg, Campath, Bexar,Erbitux, Lucentis), and (6) endocrine therapy: e.g., aromataseinhibitors (e.g., 4-hydroandrostendione, exemestane, aminoglutehimide,anastrozole, letozole), anti-estrogens (e.g., Tamoxifen, Toremifine,Raoxifene, Faslodex), steroids such as dexamethasone, (7)immuno-modulators: e.g., cytokines such as IFN-beta and IL2), inhibitorsto integrins, other adhesion proteins and matrix metalloproteinases),(8) histone deacetylase inhibitors, (9) inhibitors of signaltransduction such as inhibitors of tyrosine kinases like imatinib(Gleevec), (10) inhibitors of heat shock proteins, (11) retinoids suchas all trans retinoic acid, (12) inhibitors of growth factor receptorsor the growth factors themselves, (13) anti-mitotic compounds such asnavelbine, Paclitaxel, taxotere, vinblastine, vincristine, vindesine,and vinorelbine, (14) anti-inflammatories such as COX inhibitors and(15) cell cycle regulators, e.g., check point regulators and telomeraseinhibitors.

The term “sphingolipid” as used herein refers to the class of compoundsin the art known as sphingolipids, including, but not limited to thefollowing compounds (see http//www.lipidmaps.org as the site containingthe links indicated by the bracketed alphanumeric strings below, whichlinks contain chemical formulas, structural information, etc. for thecorresponding compounds):

Sphingoid Bases [SP01]

-   Sphing-4-enines (Sphingosines) [SP0101]-   Sphinganines [SP0102]-   4-Hydroxysphinganines (Phytosphingosines) [SP0103]-   Sphingoid base homologs and variants [SP0104]-   Sphingoid base 1-phosphates [SP0105]-   Lysosphingomyelins and lysoglycosphingolipids [SP0106]-   N-methylated sphingoid bases [SP0107]-   Sphingoid base analogs [SP0108]

Ceramides [SP02]

-   N-acylsphingosines (ceramides) [SP0201]-   N-acylsphinganines (dihydroceramides) [SP0202]-   N-acyl-4-hydroxysphinganines (phytoceramides) [SP0203]

Acylceramides [SP0204]

-   Ceramide 1-phosphates [SP0205]

Phosphosphingolipids [SP03]

-   Ceramide phosphocholines (sphingomyelins) [SP0301]-   Ceramide phosphoethanolamines [SP0302]-   Ceramide phosphoinositols [SP0303]

Phosphonosphingolipids [SP04]

Neutral glycosphingolipids [SP05]

-   Simple Glc series (GlcCer, LacCer, etc) [SP0501]-   GalNAcb1-3Gala1-4Galb1-4Glc-(Globo series) [SP0502]-   GalNAcb1-4Galb1-4Glc-(Ganglio series) [SP0503]-   Galb1-3GlcNAcb1-3Galb1-4Glc-(Lacto series) [SP0504]-   Galb1-4GlcNAcb1-3Galb1-4Glc-(Neolacto series) [SP0505]-   GalNAcb1-3Gala1-3Galb1-4Glc-(Isoglobo series) [SP0506]-   GlcNAcb1-2Mana1-3Manb1-4Glc-(Mollu series) [SP0507]-   GalNAcb1-4GlcNAcb1-3Manb1-4Glc-(Arthro series) [SP0508]-   Gal-(Gala series) [SP0509]-   Other [SP0510]

Acidic Glycosphingolipids [SP06]

-   Gangliosides [SP0601]-   Sulfoglycosphingolipids (sulfatides) [SP0602]-   Glucuronosphingolipids [SP0603]-   Phosphoglycosphingolipids [SP0604]-   Other [SP0600]

Basic Glycosphingolipids [SP07] Amphoteric Glycosphingolipids [SP08]Arsenosphingolipids [SP09]

The present invention provides anti-sphingolipid S1P agents that areuseful for treating or preventing hyperproliferative disorders such ascancer and cardiovascular or cerebrovascular diseases and disorders andvarious ocular disorders, as described in greater detail below. Inparticular the invention is drawn to S1P and its variants including butare not limited to sphingosine-1-phosphate [sphingene-1-phosphate;D-erythro-sphingosine-1-phosphate; sphing-4-enine-1-phosphate;(E,2S,3R)-2-amino-3-hydroxy-octadec-4-enoxy]phosphonic acid (AS26993-30-6), DHS1P is defined as dihydrosphingosine-1-phosphate[sphinganine-1-phosphate;[(2S,3R)-2-amino-3-hydroxy-octadecoxy]phosphonic acid;D-Erythro-dihydro-D-sphingosine-1-phosphate (CAS 19794-97-9]; SPC issphingosylphosphoryl choline, lysosphingomyelin,sphingosylphosphocholine, sphingosine phosphorylcholine, ethanaminium;2-((((2-amino-3-hydroxy-4-octadecenyl)oxy)hydroxyphosphinyl)oxy)-N,N,N-trimethyl-,chloride, (R-(R*,S*-(E))),2-[[(E,2R,3S)-2-amino-3-hydroxy-octadec-4-enoxy]-hydroxy-phosphoryl]oxyethyl-trimethyl-azaniumchloride (CAS 10216-23-6).

The term “epitope” or “antigenic determinant” when used herein, unlessindicated otherwise, refers to the region of S1P to which an anti-S1Pagent is reactive to.

The term “hyperproliferative disorder” refers to diseases and disordersassociated with, the uncontrolled proliferation cells, including but notlimited to uncontrolled growth of organ and tissue cells resulting incancers or neoplasia and benign tumors. Hyperproliferative disordersassociated with endothelial cells can result in diseases of angiogenesissuch as angiomas, endometriosis, obesity, age-related maculardegeneration and various retinopathies, as well as the proliferation ofendothelial cells and smooth muscle cells that cause restenosis as aconsequence of stenting in the treatment of atherosclerosis.Hyperproliferative disorders involving fibroblasts (for example,fibrogenesis) include but are not limited to disorders of excessivescarring (for example, fibrosis) such as age-related maculardegeneration, cardiac remodeling and failure associated with myocardialinfarction, excessive wound healing such as commonly occurs as aconsequence of surgery or injury, keloids, and fibroid tumors andstenting.

The compositions of the invention are used in methods ofsphingolipid-based therapy. “Therapy” refers to the prevention and/ortreatment of diseases, disorders or physical trauma.

The term “sphingolipid metabolite” refers to a compound from which asphingolipid is made, as well as a that results from the degradation ofa particular sphingolipid. In other words, a “sphingolipid metabolite”is a compound that is involved in the sphingolipid metabolic pathways.Metabolites include metabolic precursors and metabolic products. Theterm “metabolic precursors” refers to compounds from which sphingolipidsare made. Metabolic precursors of particular interest include but arenot limited to SPC, sphingomyelin, dihydrosphingosine, dihydroceramide,and 3-ketosphinganine. The term “metabolic products” refers to compoundsthat result from the degradation of sphingolipids, such asphosphorylcholine (e.g., phosphocholine, choline phosphate), fattyacids, including free fatty acids, and hexadecanal (e.g.,palmitaldehyde).

As used herein, the term “therapeutic” encompasses the fill spectrum oftreatments for a disease or disorder. A “therapeutic” agent of theinvention may act in a manner that is prophylactic or preventive,including those that incorporate procedures designed to targetindividuals that can be identified as being at risk (pharmacogenetics);or in a manner that is ameliorative or curative in nature; or may act toslow the rate or extent of the progression of at least one symptom of adisease or disorder being treated; or may act to minimize the timerequired, the occurrence or extent of any discomfort or pain, orphysical limitations associated with recuperation from a disease,disorder or physical trauma; or may be used as an adjuvant to othertherapies and treatments.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The term “combination therapy” refers to a therapeutic regimen thatinvolves the provision of at least two distinct therapies to achieve anindicated therapeutic effect. For example, a combination therapy mayinvolve the administration of two or more chemically distinct activeingredients, for example, a fast-acting chemotherapeutic agent and ananti-lipid antibody. Alternatively, a combination therapy may involvethe administration of an anti-lipid antibody and/or one or morechemotherapeutic agents, alone or together with the delivery of anothertreatment, such as radiation therapy and/or surgery. Further, acombination therapy may involve administration of an anti-lipid antibodytogether with one or more other biological agents (e.g., anti-VEGF,TGFβ, PDGF, or bFGF agent), chemotherapeutic agents and anothertreatment such as radiation and/or surgery. In the context of theadministration of two or more chemically distinct active ingredients, itis understood that the active ingredients may be administered as part ofthe same composition or as different compositions. When administered asseparate compositions, the compositions comprising the different activeingredients may be administered at the same or different times, by thesame or different routes, using the same of different dosing regimens,all as the particular context requires and as determined by theattending physician. Similarly, when one or more anti-lipid antibodyspecies, for example, an anti-S1P antibody, alone or in conjunction withone or more chemotherapeutic agents are combined with, for example,radiation and/or surgery, the drug(s) may be delivered before or aftersurgery or radiation treatment.

“Monotherapy” refers to a treatment regimen based on the delivery of onetherapeutically effective compound, whether administered as a singledose or several doses over time.

“Neoplasia” or “cancer” refers to abnormal and uncontrolled cell growth.A “neoplasm”, or tumor or cancer, is an abnormal, unregulated, anddisorganized proliferation of cell growth, and is generally referred toas cancer. A neoplasm may be benign or malignant. A neoplasm ismalignant, or cancerous, if it has properties of destructive growth,invasiveness, and metastasis. Invasiveness refers to the local spread ofa neoplasm by infiltration or destruction of surrounding tissue,typically breaking through the basal laminas that define the boundariesof the tissues, thereby often entering the body's circulatory system.Metastasis typically refers to the dissemination of tumor cells bylymphatics or blood vessels. Metastasis also refers to the migration oftumor cells by direct extension through serous cavities, or subarachnoidor other spaces. Through the process of metastasis, tumor cell migrationto other areas of the body establishes neoplasms in areas away from thesite of initial appearance.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” region comprises framework and CDRs (otherwise knownas hypervariables) and refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a β-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat, et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (for example, residues24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variabledomain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chainvariable domain; Kabat, et al. (1991), above) and/or those residues froma “hypervariable loop” (for example residues 26-32 (L1), 50-52 (L2), and91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55(H2), and 96-101 (H3) in the heavy chain variable domain; Chothia andLesk J. Mol. Biol. 196:901-917 (1987)). “Framework” or “FR’ residues arethose variable domain residues other than the hypervariable regionresidues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.Presently there are five major classes of immunoglobulins: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), antibody fragments, and binding agentsthat employ the CDRs (or variant thereof that retain antigen bindingactivity) of the parent antibody. Antibodies are defined herein asretaining at least one desired activity of the parent antibody. Desiredactivities can include the ability to bind the antigen specifically, theability to inhibit proleration in vitro, the ability to inhibitangiogenesis in vivo, and the ability to alter cytokine profile invitro. “Antibody fragments” comprise a portion of a full-lengthantibody, generally the antigen binding or variable domain thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, forexample, the individual antibodies comprising the population areidentical except for possible naturally occurring mutations that may bepresent in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations that typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler, et al., Nature 256:495 (1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson, et al., Nature 352:624-628 (1991) andMarks et al., J. Mol. Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones, et al., Nature 321:522-525 (1986);Reichmann, et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992) and Hansen, WO2006105062.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains that enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger, et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). The expression “linearantibodies” when used throughout this application refers to theantibodies described in Zapata, et al. Protein Eng. 8 (10):1057-1062(1995). Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair of antigen binding regions.Linear antibodies can be bispecific or monospecific.

A “variant” anti-sphingolipid antibody, refers herein to a moleculewhich differs in amino acid sequence from a “parent” anti-sphingolipidantibody amino acid sequence by virtue of addition, deletion, and/orsubstitution of one or more amino acid residue(s) in the parent antibodysequence and retains at least one desired activity of the parentanti-binding antibody. Desired activities can include the ability tobind the antigen specifically, the ability to inhibit proleration invitro, the ability to inhibit angiogenesis in vivo, and the ability toalter cytokine profile in vitro. In one embodiment, the variantcomprises one or more amino acid substitution(s) in one or morehypervariable region(s) of the parent antibody. For example, the variantmay comprise at least one, e.g. from about one to about ten, andpreferably from about two to about five, substitutions in one or morehypervariable regions of the parent antibody. Ordinarily, the variantwill have an amino acid sequence having at least 50% amino acid sequenceidentity with the parent antibody heavy or light chain variable domainsequences, more preferably at least 65%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% sequenceidentity. Identity or homology with respect to this sequence is definedherein as the percentage of amino acid residues in the candidatesequence that are identical with the parent antibody residues, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. None of N-terminal, C-terminal,or internal extensions, deletions, or insertions into the antibodysequence shall be construed as affecting sequence identity or homology.The variant retains the ability to bind a sphingolipid and preferablyhas desired activities which are superior to those of the parentantibody. For example, the variant may have a stronger binding affinity,enhanced ability to reduce angiogenesis and/or halt tumor progression.To analyze such desired properties (for example less immunogenic, longerhalf-life, enhanced stability, enhanced potency), one should compare aFab form of the variant to a Fab form of the parent antibody or a fulllength form of the variant to a full length form of the parent antibody,for example, since it has been found that the format of theanti-sphingolipid antibody impacts its activity in the biologicalactivity assays disclosed herein. The variant antibody of particularinterest herein can be one which displays at least about 5%, preferablyat least about 10%, 25%, 59%, or more of at least one desired activity.The preferred variant is one that has superior biophysical properties asmeasured in vitro or superior activities biological as measured in vitroor in vivo when compared to the parent antibody.

The “parent” antibody herein is one that is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and, if present, has humanantibody constant region(s). For example, the parent antibody may be ahumanized or human antibody.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or composition thatis detectable.

“Neuropathic pain” is a chronic pain state caused by pathologic changesin the nervous system.

A “patentable” composition, process, machine, or article of manufactureaccording to the invention means that the subject matter satisfies allstatutory requirements for patentability at the time the analysis isperformed. For example, with regard to novelty, non-obviousness, or thelike, if later investigation reveals that one or more claims encompassone or more embodiments that would negate novelty, non-obviousness,etc., the claim(s), being limited by definition to “patentable”embodiments, specifically exclude the unpatentable embodiment(s). Also,the claims appended hereto are to be interpreted both to provide thebroadest reasonable scope, as well as to preserve their validity.Furthermore, the claims are to be interpreted in a way that (1)preserves their validity and (2) provides the broadest reasonableinterpretation under the circumstances, if one or more of the statutoryrequirements for patentability are amended or if the standards changefor assessing whether a particular statutory requirement forpatentability is satisfied from the time this application is filed orissues as a patent to a time the validity of one or more of the appendedclaims is questioned.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the agents and compoundsof this invention and which are not biologically or otherwiseundesirable. In many cases, the agents and compounds of this inventionare capable of forming acid and/or base salts by virtue of the presenceof charged groups, for example, charged amino and/or carboxyl groups orgroups similar thereto. Pharmaceutically acceptable acid addition saltsmay be prepared from inorganic and organic acids, while pharmaceuticallyacceptable base addition salts can be prepared from inorganic andorganic bases. For a review of pharmaceutically acceptable salts (seeBerge, et al. (1977) J. Pharm. Sci., vol. 66, 1-19).

A “plurality” means more than one.

The terms “separated”, “purified”, “isolated”, and the like mean thatone or more components of a sample contained in a sample-holding vesselare or have been physically removed from, or diluted in the presence of,one or more other sample components present in the vessel. Samplecomponents that may be removed or diluted during a separating orpurifying step include, chemical reaction products, unreacted chemicals,proteins, carbohydrates, lipids, and unbound molecules.

The term “species” is used herein in various contexts, e.g., aparticular species of drug. In each context, the term refers to apopulation of chemically indistinct molecules of the sort referred inthe particular context.

“Specifically associate” and “specific association” and the like referto a specific, non-random interaction between two molecules, whichinteraction depends on the presence of structural,hydrophobic/hydrophilic, and/or electrostatic features that allowappropriate chemical or molecular interactions between the molecules.

A “subject” or “patient” refers to an animal in need of treatment thatcan be effected by molecules of the invention. Animals that can betreated in accordance with the invention include vertebrates, withmammals such as bovine, canine, equine, feline, ovine, porcine, andprimate (including humans and non-human primates) animals beingparticularly preferred examples.

A “therapeutically effective amount” (or “effective amount”) refers toan amount of an active ingredient, e.g., an agent according to theinvention, sufficient to effect treatment when administered to a subjector patient. Accordingly, what constitutes a therapeutically effectiveamount of a composition according to the invention may be readilydetermined by one of ordinary skill in the art. In the context of oculartherapy, a “therapeutically effective amount” is one that produces anobjectively measured change in one or more parameters associated withtreatment of the ocular disease or condition including an increase ordecrease in the expression of one or more genes correlated with theocular disease or condition, induction of apoptosis or other cell deathpathways, clinical improvement in symptoms, a decrease in aberrantneovascularization or in inflammation, etc. Of course, thetherapeutically effective amount will vary depending upon the particularsubject and condition being treated, the weight and age of the subject,the severity of the disease condition, the particular compound chosen,the dosing regimen to be followed, timing of administration, the mannerof administration and the like, all of which can readily be determinedby one of ordinary skill in the art. It will be appreciated that in thecontext of combination therapy, what constitutes a therapeuticallyeffective amount of a particular active ingredient may differ from whatconstitutes a therapeutically effective amount of the active ingredientwhen administered as a monotherapy (ie., a therapeutic regimen thatemploys only one chemical entity as the active ingredient).

The term “treatment” or “treating” of a disease or disorder includespreventing or protecting against the disease or disorder (that is,causing the clinical symptoms not to develop); inhibiting the disease ordisorder (i.e., arresting or suppressing the development of clinicalsymptoms; and/or relieving the disease or disorder (i.e., causing theregression of clinical symptoms). As will be appreciated, it is notalways possible to distinguish between “preventing” and “suppressing” adisease or disorder since the ultimate inductive event or events may beunknown or latent. Accordingly, the term “prophylaxis” will beunderstood to constitute a type of “treatment” that encompasses both“preventing” and “suppressing.” The term “treatment” thus includes“prophylaxis”.

The term “therapeutic regimen” means any treatment of a disease ordisorder using chemotherapeutic drugs, radiation therapy, surgery, genetherapy, DNA vaccines and therapy, antisense-based therapies includingsiRNA therapy, anti-angiogenic therapy, immunotherapy, bone marrowtransplants, aptamers and other biologics such as antibodies andantibody variants, receptor decoys and other protein-based therapeutics.

SUMMARY OF THE INVENTION

The present application describes patentable methods of treating orpreventing pain, which methods comprise administering to a subject,including a human subject, having or believed to be at risk of havingpain, an antibody (or an antibody fragment or derivative) that binds andneutralizes S1P. The antibody may be a polyclonal or monoclonalantibody, or an antibody fragment or derivative, that retains bindingability for S1P. Preferred are humanized monoclonal antibodies orfragments thereof that bind S1P.

The instant methods are particularly useful for treating or preventingpain such as neuropathic pain, allodynia and hyperalgesia. Treatment ofpain associated with chemotherapeutic drug treatment is one embodimentof the invention.

These and other aspects and embodiments of the invention are discussedin greater detail in the sections that follow. The foregoing and otheraspects of the invention will become more apparent from the followingdetailed description, accompanying drawings, and the claims. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. In addition, thematerials, methods, and examples below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

A brief summary of each of the figures is provided below.

FIG. 1. Anti-S1P antibody LT1002 but not control antibody blockspaclitaxel-induced neuropathic pain. FIG. 1 is a line graph showingthat, when compared to the vehicle group (□), administration ofpaclitaxel () led to a time-dependent development of mechano-allodynia,which was significantly attenuated at 16 h by intravenous delivery ofLT1002 (▴), but not by control antibody LT1017 (▾). Results areexpressed as mean±SEM. Behavioral data for 3 animals was analyzed bytwo-tailed, two-way ANOVA with Bonferroni post hoc comparisons to thepaclitaxel group where *P<0.01 and **P<0.001 for paclitaxel vs vehicleand †P<0.05 for paclitaxel+LT1002 vs paclitaxel.

FIG. 2. Anti-S1P antibody LT1002 but not control antibody blocksceramide-induced hyperalgesia. FIG. 2 is a line graph showing that whenwhen compared to rats given intraplantar injection of saline (antibodyvehicle, Veh, Δ, n=3), an intraplantar injection of ceramide (10 ug, ⋄,n=3) led to a time-dependent development of thermal hyperalgesia thatwas attenuated by the anti-S1P antibody LT1002 (242 ug, , n=3) but notby isotype control antibody LT1017 (286 ug, ▪, n=3). Given alone, LT1002(∘, n=3) or LT1017 (□, n=3) had no effect.

DETAILED DESCRIPTION OF THE INVENTION Compounds

The present invention describes certain anti-S1P agents, particularlythose that are immune-derived moieties, including antibodies, which arespecifically reactive with the bioactive lipid S1P; in other words, thebioactive lipid to which the anti-S1P agent reacts is S1P.

Antibody molecules or immunoglobulins are large glycoprotein moleculeswith a molecular weight of approximately 150 kDa, usually composed oftwo different kinds of polypeptide chain. One polypeptide chain, termedthe “heavy” chain (H) is approximately 50 kDa. The other polypeptide,termed the “light” chain (L), is approximately 25 kDa. Eachimmunoglobulin molecule usually consists of two heavy chains and twolight chains. The two heavy chains are linked to each other by disulfidebonds, the number of which varies between the heavy chains of differentimmunoglobulin isotypes. Each light chain is linked to a heavy chain byone covalent disulfide bond. In any given naturally occurring antibodymolecule, the two heavy chains and the two light chains are identical,harboring two identical antigen-binding sites, and are thus said to bedivalent, i.e., having the capacity to bind simultaneously to twoidentical molecules.

The “light” chains of antibody molecules from any vertebrate species canbe assigned to one of two clearly distinct types, kappa (k) and lambda(l), based on the amino acid sequences of their constant domains. Theratio of the two types of light chain varies from species to species. Asa way of example, the average k to l ratio is 20:1 in mice, whereas inhumans it is 2:1 and in cattle it is 1:20.

The “heavy” chains of antibody molecules from any vertebrate species canbe assigned to one of five clearly distinct types, called isotypes,based on the amino acid sequences of their constant domains. Someisotypes have several subtypes. The five major classes of immunoglobulinare immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G(IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is themost abundant isotype and has several subclasses (IgG1, 2, 3, and 4 inhumans). The Fc fragment and hinge regions differ in antibodies ofdifferent isotypes, thus determining their functional properties.However, the overall organization of the domains is similar in allisotypes.

The term “variable region” refers to the N-terminal portion of theantibody molecule or a fragment thereof. In general, each of the fourchains has a variable (V) region in its amino terminal portion, whichcontributes to the antigen-binding site, and a constant (C) region,which determines the isotype. The light chains are bound to the heavychains by many noncovalent interactions and by disulfide bonds and the Vregions of the heavy and light chains pair in each arm of antibodymolecule to generate two identical antigen-binding sites. Some aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains [see Kabat, et al. (1991), Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. and Clothia et al. (1985), J. Mol. Biol, vol 186:651].

Of note, variability is not uniformly distributed throughout thevariable domains of antibodies, but is concentrated in three segmentscalled “complementarity-determining regions” (CDRs) or “hypervariableregions” both in the light-chain and the heavy-chain variable domains.The more highly conserved portions of variable domains are called the“framework region” (FR). The variable domains of native heavy and lightchains each comprise four FR regions connected by three CDRs. The CDRsin each chain are held together in close proximity by the FR regionsand, with the CDRs from the other chains, form the antigen-binding siteof antibodies [see Kabat, et al. (1991), Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md.]. Collectively, the 6 CDRs contribute to the bindingproperties of the antibody molecule for the antigen. However, even asingle variable domain (or half of an Fv, comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen[see Pluckthun (1994), in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315].

The term “constant domain” refers to the C-terminal region of anantibody heavy or light chain. Generally, the constant domains are notdirectly involved in the binding properties of an antibody molecule toan antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.Here, “effector functions” refer to the different physiological effectsof antibodies (e.g., opsonization, cell lysis, mast cell, basophil andeosinophil degranulation, and other processes) mediated by therecruitment of immune cells by the molecular interaction between the Fcdomain and proteins of the immune system. The isotype of the heavy chaindetermines the functional properties of the antibody. Their distinctivefunctional properties are conferred by the carboxy-terminal portions ofthe heavy chains, where they are not associated with light chains.

As used herein, “antibody fragment” refers to a portion of an intactantibody that includes the antigen binding site or variable regions ofan intact antibody, wherein the portion can be free of the constantheavy chain domains (e.g., CH2, CH3, and CH4) of the Fc region of theintact antibody. Alternatively, portions of the constant heavy chaindomains (e.g., CH2, CH3, and CH4) can be included in the “antibodyfragment”. Examples of antibody fragments are those that retainantigen-binding and include Fab, Fab′, F(ab′)2, Fd, and Fv fragments;diabodies; triabodies; single-chain antibody molecules (sc-Fv);minibodies, nanobodies, and multispecific antibodies formed fromantibody fragments. By way of example, a Fab fragment also contains theconstant domain of a light chain and the first constant domain (CH1) ofa heavy chain.

The term “variant” refers to an amino acid sequence which differs fromthe native amino acid sequence of an antibody by at least one amino acidresidue or modification. A native or parent or wild-type amino acidsequence refers to the amino acid sequence of an antibody found innature. “Variant” of the antibody molecule includes, but is not limitedto, changes within a variable region or a constant region of a lightchain and/or a heavy chain, including the hypervariable or CDR region,the Fc region, the Fab region, the CH1 domain, the CH2 domain, the CH3domain, and the hinge region.

The term “specific” refers to the selective binding of an antibody toits target epitope. Antibody molecules can be tested for specificity ofbinding by comparing binding of the antibody to the desired antigen tobinding of the antibody to unrelated antigen or analogue antigen orantigen mixture under a given set of conditions. Preferably, an antibodyaccording to the invention will lack significant binding to unrelatedantigens, or even analogs of the target antigen. Here, the term“antigen” refers to a molecule that is recognized and bound by anantibody molecule or immune-derived moiety that binds to the antigen.The specific portion of an antigen that is bound by an antibody istermed the “epitope.” A “hapten” refers to a small molecule that can,under most circumstances, elicit an immune response (i.e., act as anantigen) only when attached to a carrier molecule, for example, aprotein, polyethylene glycol (PEG), colloidal gold, silicone beads, andthe like. The carrier may be one that also does not elicit an immuneresponse by itself.

The term “antibody” is used in the broadest sense, and encompassesmonoclonal, polyclonal, multispecific (e.g., bispecific, wherein eacharm of the antibody is reactive with a different epitope or the same ordifferent antigen), minibody, heteroconjugate, diabody, triabody,chimeric, and synthetic antibodies, as well as antibody fragments thatspecifically bind an antigen with a desired binding property and/orbiological activity.

The term “monoclonal antibody” (mAb) refers to an antibody, orpopulation of like antibodies, obtained from a population ofsubstantially homogeneous antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, monoclonal antibodies can be made by the hybridoma method firstdescribed by Kohler and Milstein (1975), Nature, vol 256: 495-497, or byrecombinant DNA methods.

The term “chimeric” antibody (or immunoglobulin) refers to a moleculecomprising a heavy and/or light chain which is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity [Cabilly et al. (1984), infra; Morrison et al.,Proc. Natl. Acad. Sci. U.S.A. 81:6851].

The term “humanized antibody” refers to forms of antibodies that containsequences from non-human (eg, murine) antibodies as well as humanantibodies. A humanized antibody can include conservative amino acidsubstitutions or non-natural residues from the same or different speciesthat do not significantly alter its binding and/or biologic activity.Such antibodies are chimeric antibodies that contain minimal sequencederived from non-human immunoglobulins. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary-determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, camel, bovine, goat, or rabbit having thedesired properties. Furthermore, humanized antibodies can compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and maximize antibody performance. Thus, in general, ahumanized antibody will comprise all of at least one, and in one aspecttwo, variable domains, in which all or all of the hypervariable loopscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), or that of a humanimmunoglobulin. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567;Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat.No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger,M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No.0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European PatentNo. 0,239,400 B1; Padlan, E. A. et al., European Patent Application No.0,519,596 A1; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA, vol86:10029-10033).

The term “bispecific antibody” can refer to an antibody, or a monoclonalantibody, having binding properties for at least two different epitopes.In one embodiment, the epitopes are from the same antigen. In anotherembodiment, the epitopes are from two different antigens. Methods formaking bispecific antibodies are known in the art. For example,bispecific antibodies can be produced recombinantly using theco-expression of two immunoglobulin heavy chain/light chain pairs.Alternatively, bispecific antibodies can be prepared using chemicallinkage. Bispecific antibodies include bispecific antibody fragments.

The term “heteroconjugate antibody” can refer to two covalently joinedantibodies. Such antibodies can be prepared using known methods insynthetic protein chemistry, including using crosslinking agents. Asused herein, the term “conjugate” refers to molecules formed by thecovalent attachment of one or more antibody fragment(s) or bindingmoieties to one or more polymer molecule(s).

The term “biologically active” refers to an antibody or antibodyfragment that is capable of binding the desired epitope and in some wayexerting a biologic effect. Biological effects include, but are notlimited to, the modulation of a growth signal, the modulation of ananti-apoptotic signal, the modulation of an apoptotic signal, themodulation of the effector function cascade, and modulation of otherligand interactions.

The term “recombinant DNA” refers to nucleic acids and gene productsexpressed therefrom that have been engineered, created, or modified byman. “Recombinant” polypeptides or proteins are polypeptides or proteinsproduced by recombinant DNA techniques, for example, from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or proteins are thoseprepared by chemical synthesis.

The term “expression cassette” refers to a nucleotide molecule capableof effecting expression of a structural gene (i.e., a protein codingsequence, such as an antibody of the invention) in a host compatiblewith such sequences. Expression cassettes include at least a promoteroperably linked with the polypeptide-coding sequence, and, optionally,with other sequences, e.g., transcription termination signals.Additional regulatory elements necessary or helpful in effectingexpression may also be used, e.g., enhancers. Thus, expression cassettesinclude plasmids, expression vectors, recombinant viruses, any form ofrecombinant “naked DNA” vector, and the like.

Sources of antibody are not limited to those exemplified herein (e.g.,murine and humanized murine antibody). Antibodies may be raised in manyspecies including mammalian species (for example, mouse, rat, camel,bovine, goat, horse, guinea pig, hamster, sheep and rabbit) and birds(duck, chicken). Antibodies raised may derive from a different speciesfrom the animal in which they are raised. For example, the XenoMouse™(Abgenix, Inc., Fremont Calif.) produces fully human monoclonalantibodies. For certain purposes, native human antibodies, such asautoantibodies to S1P isolated from individuals who may show a titer ofsuch S1P autoantibody may be used. Alternatively, a human antibodysequence library may be used to generate antibodies comprising a humansequence.

Antibody Generation and Characterization

Generation of anti-S1P antibodies, humanized anti-S1P antibodies andvariants thereof are described, e.g., in U.S. Pat. No. 8,026,342 whichis commonly owned with the instant application and is incorporatedherein by reference in its entirety. Methods for determining antibodyare also described therein. Preferred humanized or variant antibodiesare those which bind S1P with a K_(d) value of no more than about 1×10⁻⁷M, preferably no more than about 1×10⁻⁸ M, and most preferably no morethan about 5×10⁻⁹ M. Sequences of several preferred anti-S1P antibodiesare shown in U.S. Pat. No. 8,026,342.

Aside from antibodies with strong binding affinity for S1P, it is alsodesirable to select humanized or variant antibodies that have otherbeneficial properties from a therapeutic perspective. For example, theantibody may be one that reduces inflammation.

A preferred formulation for systemic administration of the anti-S1Pantibodies is disclosed in provisional patent application U.S.61/042,736, “Pharmaceutical Compositions for BindingSphingosine-1-Phosphate”, filed Apr. 5, 2008, and commonly owned withthe instant invention, which is incorporated herein in its entirety.

Applications

The invention is drawn to compositions and methods for treating orpreventing pain, using one or more therapeutic agents, e.g., antibodies,that bind S1P. These therapeutic methods and compositions act bychanging the effective concentration, i.e., the absolute, relative,effective and/or available concentration and/or activities, of certainundesired bioactive lipids. Lowering the effective concentration of thebioactive lipid may be said to “neutralize” the target lipid or itsundesired effects, including downstream effects. Here, “undesired”refers to a bioactive lipid that is unwanted due to its involvement in adisease process, for example, as a signaling molecule, or to an unwantedamount of a bioactive lipid which contributes to disease when present inexcess.

Without wishing to be bound by any particular theory, it is believedthat inappropriate concentrations of S1P and/or its metabolites ordownstream effectors, may cause or contribute to the development ofvarious diseases and disorders. As such, the compositions and methodscan be used to treat these diseases and disorders, particularly bydecreasing the effective in vivo concentration of S1P. In particular, itis believed that the compositions and methods of the invention areuseful in treating and/or preventing pain, including neuropathic painand inflammatory pain. It will be appreciated that many diseases andconditions are characterized, at least in part, by multiple pathologicalprocesses and that the classifications provided herein are fordescriptive convenience and do not limit the invention.

Antibodies as Drugs

The use of monoclonal antibodies (mAbs) as a therapeutic treatment for avariety of diseases and disorders is rapidly increasing because theyhave been shown to be safe and efficacious therapeutic agents. Approvedtherapeutic mAbs include Avastin®, Erbitux®, and Rituxan®. AdditionalmAbs are in various phases of clinical development for a variety ofdiseases with the majority targeting various forms of cancer. Ingeneral, monoclonal antibodies are generated in non-human mammals. Thetherapeutic utility of murine monoclonal antibodies is limited, however,principally due to the fact that human patients mount their own antibodyresponse to murine antibodies. This response, the so-called HAMA (humananti-mouse antibody) response, results in the eventual neutralizationand rapid elimination of murine mAbs. This limitation has been overcomewith the development of a process called “humanization” of murineantibodies. Humanization greatly lessens the development of an immuneresponse against the administered therapeutic MAb and thereby avoids thereduction of half-life and therapeutic efficacy consequent on HAMA. Forthe most part, the humanization process consists of grafting the murinecomplementary determining regions (CDRs) into the framework region (FR)of a human immunoglobulin. This strategy is referred to as “CDRgrafting”. “Backmutation” to murine amino acid residues of selectedresidues in the human FR is often required to regain affinity that islost in the initial grafted construct.

The manufacture of mAbs is a complex process that stems from thevariability of the protein itself. The variability of mAbs can belocalized to the protein backbone and/or to the carbohydrate moiety. Theheterogeneity can be attributed to the formation of alternativedisulfide pairings, deamidation and the formation of isoaspartylresidues, methionine and cysteine oxidation, cyclization of N-terminalglutamine residues to pyroglutamate and partial enzymatic cleavage ofC-terminal lysines by mammalian carboxypeptidases. Engineering iscommonly applied to antibody molecules to improve their properties, suchas enhanced stability, resistance to proteases, aggregation behavior andenhance the expression level in heterologous systems.

The murine anti-S1P mAb was humanized by grafting the six CDRs fromLpath's murine anti-S1P antibody, LT1002 (sphingomab), into a humanframework. Further modifications were engineered to further refine andoptimize the antibody performance. The humanized mAb, LT1009(sonepcizumab) presented the same characteristics as the LT1002 and isthus suitable for testing in clinical trials.

One way to control the amount of undesirable sphingolipids, e.g.,undesired levels of S1P, in a patient is by providing a composition thatcomprises one or more humanized anti-sphingolipid antibodies to bind oneor more sphingolipids, thereby acting as therapeutic “sponges” thatreduce the level of free undesirable sphingolipids. When a compound isreferred to as “free”, the compound is not in any way restricted fromreaching the site or sites where it exerts its undesirable effects.Typically, a free compound is present in blood and tissue, which eitheris or contains the site(s) of action of the free compound, or from whicha compound can freely migrate to its site(s) of action. A free compoundmay also be available to be acted upon by any enzyme that converts thecompound into an undesirable compound.

S1P and Pain

S1P has been associated with a number of diseases and conditions, suchas cancer, cardiovascular disease, aberrant angiogenesis, inflammationand pain. The roles of S1P in disease are summarized, for example, inU.S. Pat. No. 8,026,342 which is commonly owned with the instantinvention and incorporated by reference herein in its entirety.

Pain is the most common reason for doctor visits in the US and ispresent as part of a broad spectrum of diseases, disorders andconditions. Pain may be acute or chronic and may be classified accordingto location in the body and/or by etiology, although in many cases theetiology of pain is not understood or may be due to several possiblecauses, which may overlap. Pain may also be described qualitatively, asallodynia (abnormal sensory perception of pain) or hyperalgesia(exaggerated pain sensations), for example.

Neuropathic pain is a complex, often chronic form of pain associatedwith damage or dysfunction of the nervous system. Simply stated,neuropathic pain is a chronic pain state caused by pathological changesin the nervous system. Myers, et al (2006) Drug Disc. Today 11: 8-20.Causes of acute and/or chronic neuropathic pain include, but are notlimited to, injury, trauma, or damage to the central or peripheralnervous system (e.g., spinal cord injury, disc herniation, multiplesclerosis or other degenerative or neurodegenerative disease),inflammation, drug exposure (for example, cytotoxics such as paclitaxel(TAXOL), cisplatin, and other chemotherapeutic agents), diabetes, viraldisease (such as, for example, HIV and herpes zoster), metabolicdisease, severe ischemic insults, nutrient deficiency, toxin exposure,and cancer. Cancer neuropathic pain may result directly from tumorimpingement on nerves, or indirectly such as from radiation, surgery, ordrug treatment. Neuropathic pain is mediated through neuroinflammatorymechanisms controlled by inflammatory responses to the initial insultand affecting nervous system tissue. Myers, et al (2006), Drug Disc.Today 11: 8-20. Many inflammatory mediators, such as TNFα, have beenfound to be pivotal in neuropathic pain. Leung L, Cahill C M. (2010) JNeuroinflamm., 7:27. Neuropathic pain is unresponsive to most commonpainkillers.

Pain associated with chemotherapy is a major dose-limiting toxicity ofmany small molecule chemotherapeutic agents, particularly thecytotoxics. For instance, paclitaxel (TAXOL), an anti-neoplastic agentderived from the Pacific yew tree Taxus brevifolia, is used to treat avariety of cancers, including ovarian, breast, and non-small cell lungcancer. paclitaxel's effectiveness, however, is limited by the highlyincidental development of severe painful peripheral neuropathy such asnumbness and burning pain. An antibody against a bioactive lipidcorrelated with such pain, for example, S1P (or a derivative of such anantibody that contains a lipid-binding portion thereof), could beadministered in combination with paclitaxel in order to reduce the painassociated with the chemotherapeutic agent. As a result of amelioratingthis dose-limiting toxicity, the amount of paclitaxel to be administeredcould be even higher (and thus even more effective) when used incombination with such a monoclonal antibody or antibody derivative. Insome embodiments, the chemotherapeutic agent (or other drug) could beconjugated to or otherwise associated with the antibody or antibodyderivative, for example, by covalently linking the small moleculechemotherapeutic agent to the antibody, by linking the small moleculechemotherapeutic to a multivalent scaffold to which is also linked amonoclonal antibody or at least one bioactive lipid binding domainderived from a monoclonal antibody specifically reactive against thetarget bioactive lipid, etc.

Diabetic neuropathy in type 1 and 2 diabetes in both humans and animalmodels is characterized by pathophysiological changes in all components(sensory, motor and autonomic) of the peripheral nervous system. Inaddition to the changes of primary afferent nerves, centralsensitization is believed to be an important mechanism underlyingpersistent pain, including neuropathic and inflammatory pain. G.Baranauskas, A. Nistri (1998) Prog Neurobiol 54, 349; T. J. Coderre, R.Melzack (1992) J Neurosci 12, 3665; R. Dubner, M. A. Ruda (1992) TrendsNeurosci 15, 96; M. J. Millan (1999) Prog Neurobiol 57, 1; C. J. Woolf,S. W. Thompson, (1991) Pain 44, 293.

One of the key features of inflammatory states is that normallyinnocuous stimuli produce pain. This pain is often referred to as“inflammatory pain.” Pain arising from inflamed or injured tissues mayarise spontaneously in the absence of an external trigger.Alternatively, responses to noxious stimuli may be enhanced(hyperalgesia) or normally innocuous stimuli may produce pain(allodynia).

Inflammatory mediators are involved in the genesis, persistence, andseverity of pain. IL-6 is a potent pain-generating inflammatorymediator. IL-6 is produced in the rat spinal cord following peripheralnerve injury, with levels of IL-6 levels correlating directly with theintensity of allodynia. Arruda, et al. (2000), Brain Res. 879:216-25.IL-6 levels increase during stress or inflammation, and rheumatoidarthritis is associated with increased levels of IL-6 in synovial fluid.Matsumoto, et al (2006), Rheumatol. Int. 26:1096-1100; Desgeorges, etal. (1997), J. Rheumatol. 24:1510-1516. Neuropathic pain is prevented inIL-6 knockout mice. Xu, et al (1997), Cytokine 9:1028-1033.

IL-8 is a pain-generating inflammatory mediator. Drug treatment ofpost-herpetic neuralgia showed a decrease of 50% in IL-8 concentrations,and this decrease correlated with pain relief. Kotani, et al. (2000),New Engl. J. Med. 343:1514-1519.

TNF-α induces axonal damage, macrophage recruitment and ectopic activityin peripheral nerve fibers and plays a role in the generation ofhyperalgesia. TNFα is upregulated at the site of peripheral nervelesions and in patients with neuropathic pain. Thalidomide, a selectiveblocker of TNF production, reduces hyperalgesia in an animal model ofneuropathic pain (chronic constriction injury). George, et al. (2000),Pain 88:267-275.

A significant role of S1P in the development of pain was establishedusing various pharmacological and genetic approaches. S1P is part of theceramide metabolic pathway; ceramide is a potent proinflammatorysphingolipid which can be metabolized by ceramidase to sphingosine whichcan be converted to S1P by sphingosine kinase. As reviewed in Doyle etal. (2011) [Neurosci Lett. 499:4-8], ceramide has a well establishedrole in inflammation, and experimental data also point to a role forceramide in peripheral sensitization and both mechanical and thermalhyperalgesia. Doyle et al (2011) FASEB J. 25: 2782-2791; Joseph andLevine (2004) Eur. J. Neurosci. 20:2896-2902. Furthermore,TNF-alpha-mediated peripheral sensitization and nerve growthfactor-induced sensitization of sensory neurons also involves ceramide.Zhang et al (2002) J. Physiol. 544:385-402. S1P also contributes toexcitation of sensory neurons both in vitro and in vivo. Zhang et al.(2006) J. Physiol 575:101-113; Zhang et al (2006) J. Neurophysiol.96:1042-1052; Zhang et al. (2002) J. Physiol. 544:385-402. Doyle et al.(2011) [Pain 152:643-648] have shown that intraplantar injection of S1Pin rats led to the development of hyperalgesia. This effect is abrogatedby genetic deletion of S1PR₁ (S1P receptor1) in neurons. Mair et al(2011) PLoS One 6:e17268.

Diabetic neuropathy in type 1 and 2 diabetes in both humans and animalmodels is characterized by pathophysiological changes in all components(sensory, motor and autonomic) of the peripheral nervous system. Inaddition to the changes of primary afferent nerves, centralsensitization is believed to be an important mechanism underlyingpersistent pain, including neuropathic and inflammatory pain. G.Baranauskas, A. Nistri (1998) Prog Neurobiol 54, 349; T. J. Coderre, R.Melzack (1992) J Neurosci 12, 3665; R. Dubner, M. A. Ruda (1992) TrendsNeurosci 15, 96; M. J. Millan (1999) Prog Neurobiol 57, 1; C. J. Woolf,S. W. Thompson, (1991) Pain 44, 293.

Formulations

Anti-sphingolipid antibodies may be formulated in a pharmaceuticalcomposition that are useful for a variety of purposes, including thetreatment of diseases, disorders or physical trauma. Pharmaceuticalcompositions comprising one or more humanized anti-sphingolipidantibodies of the invention may be incorporated into kits and medicaldevices for such treatment. Medical devices may be used to administerthe pharmaceutical compositions of the invention to a patient in needthereof, and according to one embodiment of the invention, kits areprovided that include such devices. Such devices and kits may bedesigned for routine administration, including self-administration, ofthe pharmaceutical compositions of the invention. Such devices and kitsmay also be designed for emergency use, for example, in ambulances oremergency rooms, or during surgery, or in activities where injury ispossible but where full medical attention may not be immediatelyforthcoming (for example, hiking and camping, or combat situations).

Methods and Routes of Administration

The treatment for diseases and conditions discussed herein can beachieved by administering agents and compositions of the invention byvarious routes employing different formulations and devices. Suitablepharmaceutically acceptable diluents, carriers, and excipients are wellknown in the art.

One skilled in the art will appreciate that the amounts to beadministered for any particular treatment protocol can readily bedetermined. Suitable amounts might be expected to fall within the rangeof 10 μg/dose to 10 g/dose, preferably within 10 mg/dose to 1 g/dose.

Drug substances may be administered by techniques known in the art,including but not limited to systemic, subcutaneous, intradermal,mucosal, including by inhalation, and topical administration. The mucosarefers to the epithelial tissue that lines the internal cavities of thebody. For example, the mucosa comprises the alimentary canal, includingthe mouth, esophagus, stomach, intestines, and anus; the respiratorytract, including the nasal passages, trachea, bronchi, and lungs; andthe genitalia. For the purpose of this specification, the mucosa alsoincludes the external surface of the eye, i.e., the cornea andconjunctiva. Local administration (as opposed to systemicadministration) may be advantageous because this approach can limitpotential systemic side effects, but still allow therapeutic effect.

Pharmaceutical compositions used in the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations used in the present invention may beprepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). Preferred carriers include those that arepharmaceutically acceptable, particularly when the composition isintended for therapeutic use in humans. For non-human therapeuticapplications (e.g., in the treatment of companion animals, livestock,fish, or poultry), veterinarily acceptable carriers may be employed. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment the pharmaceutical compositions may be formulated andused as foams. Pharmaceutical foams include formulations such as, butnot limited to, emulsions, microemulsions, creams, jellies, andliposomes.

While basically similar in nature these formulations vary in thecomponents and the consistency of the final product. The know-how on thepreparation of such compositions and formulations is generally known tothose skilled in the pharmaceutical and formulation arts and may beapplied to the formulation of the compositions of the present invention.

Therapeutic Uses

For therapeutic applications, the anti-sphingolipid antibodies of theinvention are administered to a mammal, preferably a human, in apharmaceutically acceptable dosage form such as those discussed above,including those that may be administered to a human intravenously as abolus or by continuous infusion over a period of time, by intramuscular,intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 ug/kg toabout 50 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily or weekly dosage might range from about 1 μg/kg to about 20 mg/kgor more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays, including, for example, radiographic imaging.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for those purposes, such as conventional analgesicsand drugs such as tricyclic antidepressants and anticonvulsants (e.g.,gabapentin), which are sometimes administered for relief of neuropathicpain, for example. Such other agents may be present in the compositionbeing administered or may be administered separately. The antibody issuitably administered serially or in combination with the other agent.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the anti-sphingolipidantibody. The label on, or associated with, the container indicates thatthe composition is used for treating the condition of choice. Thearticle of manufacture may further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The invention will be better understood by reference to the followingExamples, which are intended to merely illustrate the best mode nowknown for practicing the invention. The scope of the invention is not tobe considered limited thereto.

EXAMPLES Example 1 Antibody to S1P Reduces Paclitaxel-InducedNeuropathic Pain

Paclitaxel-induced neuropathic pain and drug administration: The wellcharacterized rat model developed by Bennett was used, in which repeatedintraperitoneal (i.p) injections of low doses of paclitaxel induceneuropathic pain (mechano-allodynia) with little systemic toxicity ormotor impairment. Flatters, S. J. & Bennett, G. J. (2006) Pain 122,245-257; Jin, H. W. et al. (2008) Exp Neurol 210, 229-237; Polomano, R.C., et al. (2001) Pain 94, 293-304.

The behavioral responses last for several weeks to months, thus modelingpainful neuropathies in patients (ibid). Male Sprague Dawley rats(200-210 g starting weight) from Harlan (Indianapolis, Ind.) were housed3-4 per cage in a controlled environment (12 h light/dark cycle) withfood and water available ad libitum. All experiments were performed inaccordance with the International Association for the Study of Pain andthe National Institutes of Health guidelines on laboratory animalwelfare and the recommendations by Saint Louis University InstitutionalAnimal Care and Use Committee. All experiments were conducted with theexperimenters blinded to treatment conditions.

Paclitaxel or its vehicle (Cremophor EL and 95% dehydrated ethanol in1:1 ratio) was injected i.p in rats on four alternate days that is day(D) 0, 2, 4 and 6 at 1 mg/kg on with a final cumulative dose of 4mg/kg.1-3 The following experimental test substances were used: LT1002,and its isotype control, LT1017; these were dissolved in saline andprovided by Lpath in individual vials. Experimental test substances weregiven intravenously (i.v) at 25 mg/kg according to a dosing regimendesigned by Lpath as follows (and see experimental design, schematic inpower point format). Experimental test substances were given one daybefore (D−1) the first injection of paclitaxel, and subsequently on D2,D5, D8, D11 and D14 after the first injection of paclitaxel. Vehiclethat was used to dissolve test substance (saline) was injected accordingto the same dosing paradigm in paclitaxel-treated group or itsrespective vehicle. If injections of experimental test substancescoincided with the injection of paclitaxel (i.e. D2), experimental testsubstances were delivered 15 min before paclitaxel. Mechanicalwithdrawal thresholds were assessed with an electronic version of thevon Frey test (dynamic plantar aesthesiometer, model 37450; Ugo Basile,Milan, Italy) on D−1 before experimental test substance injection, theday after (D0) and before the first i.p. injection of paclitaxel andsubsequently on D12 and D16. To this end, each rat was placed in aPlexiglas chamber (28×40×35-cm, wire mesh floor) and allowed toacclimate for fifteen minutes. After acclimation, a servo-controlledmechanical stimulus (a pointed metallic filament) was applied to theplantar surface, which exerts a progressively increasing punctatepressure, reaching up to 50 g within 10 s. The pressure evoking a clearvoluntary hind-paw withdrawal response was recorded automatically andtaken as the mechanical threshold index. Mechanical threshold isassessed three times at each time point to yield a mean value, which isreported as mean absolute threshold (grams, g). The development ofmechano-allodynia is evidenced by a significant (P<0.05) reduction inmechanical mean absolute paw-withdrawal thresholds (grams, g) at forcesthat failed to elicit withdrawal responses before paclitaxel treatment(baseline). Paclitaxel treatments results in bilateral allodynia (ibid).Because thresholds did not differ between left and right hind paws atany time point in any group, values from both paws were averaged forfurther analysis and data presentation. A total of six groups were usedwith n=3 rats/group.

-   Group 1: Vehicle instead of paclitaxel+saline-   Group 2: Paclitaxel+saline-   Group 3: Paclitaxel+LT 1002-   Group 4: Paclitaxel+LT 1017

Paw withdrawal threshold (g) for groups 1-4 on (D−1) and before i.vinjection of experimental test substances or their vehicle (saline) were(mean+/s.em): 43.6±0.296, 43.3±0.376, 43.7±0.333, and 42.9±0.219,respectively.

Statistical Analysis. Data are expressed as mean±SEM for 3 animals pergroup and analyzed by two-tailed, two-way ANOVA with Bonferroni post hoccomparisons to the paclitaxel group. *P<0.05, **P<0.001 paclitaxel vs.vehicle group.

Results Effects of LT1002 and LT1017 on Paclitaxel-Induced NeuropathicVain

When compared to the vehicle treated group, administration of paclitaxelled to the development of mechano-allodynia (FIG. 1). The development ofmechano-allodynia at 16 h was significantly attenuated by LT1002, butnot by LT1017 (FIG. 1). Paclitaxel-induced neuropathic pain and drugadministration: The well characterized rat model developed by Bennett inwhich repeated intraperitoneal (i.p) injections of low doses ofpaclitaxel induce neuropathic pain (mechano-allodynia) with littlesystemic toxicity or motor impairment. The behavioral responses last forseveral weeks to months, thus modeling painful neuropathies in patients.Paclitaxel or its vehicle (Cremophor EL and 95% dehydrated ethanol in1:1 ratio) was injected i.p in rats on four alternate days that is day(D) 0, 2, 4 and 6 at 1 mg/kg on with a final cumulative dose of 4mg/kg.1-3 The following experimental test substances were used: LT1002and LT1017; these were dissolved in saline and provided by Lpath inindividual vials. Experimental test substances were given intravenously(i.v) at 25 mg/kg according to a dosing regimen designed by Lpath asfollows (and see experimental design, schematic in power point format).Experimental test substances were given one day before (D−1) the firstinjection of paclitaxel, and subsequently on D2, D5, D8, D11 and D14after the first injection of paclitaxel. Vehicle that was used todissolve test substance (saline) was injected according to the samedosing paradigm in paclitaxel-treated group or its respective vehicle.If injections of experimental test substances coincided with theinjection of paclitaxel (i.e. D2), experimental test substances weredelivered 15 min before paclitaxel. Mechanical withdrawal thresholdswere assessed with an electronic version of the von Frey test (dynamicplantar aesthesiometer, model 37450; Ugo Basile, Milan, Italy) on D−1before experimental test substance injection, the day after (D0) andbefore the first i.p. injection of paclitaxel and subsequently on D12and D16. To this end, each rat was placed in a Plexiglas chamber(28×40×35-cm, wire mesh floor) and allowed to acclimate for fifteenminutes. After acclimation, a servo-controlled mechanical stimulus (apointed metallic filament) was applied to the plantar surface, whichexerts a progressively increasing punctate pressure, reaching up to 50 gwithin 10 s. The pressure evoking a clear voluntary hind-paw withdrawalresponse was recorded automatically and taken as the mechanicalthreshold index. Mechanical threshold is assessed three times at eachtime point to yield a mean value, which is reported as mean absolutethreshold (grams, g). The development of mechano-allodynia is evidencedby a significant (P<0.05) reduction in mechanical mean absolutepaw-withdrawal thresholds (grams, g) at forces that failed to elicitwithdrawal responses before paclitaxel treatment (baseline). Paclitaxeltreatments results in bilateral allodynia (ibid). Because thresholds didnot differ between left and right hind paws at any time point in anygroup, values from both paws were averaged for further analysis and datapresentation. A total of six groups were used with n=3 rats/group.

-   Group 1: Vehicle instead of paclitaxel+saline-   Group 2: Paclitaxel+saline-   Group 3: Paclitaxel+LT1002-   Group 4: Paclitaxel+LT1017

Paw withdrawal threshold (g) for groups 1-6 on (D−1) and before i.vinjection of experimental test substances or their vehicle (saline) were(mean+/s.em): 43.6±0.296, 43.3±0.376, 43.7±0.333 and 42.9±0.219,respectively.

Statistical Analysis. Data are expressed as mean ±SEM for 3 animals pergroup and analyzed by two-tailed, two-way ANOVA with Bonferroni post hoccomparisons to the paclitaxel group. *P<0.05, **P<0.001 paclitaxel vs.vehicle group.

Results

Effects of LT1002 and LT1017 on paclitaxel-induced neuropathic pain.When compared to the vehicle treated group, administration of paclitaxelled to the development of mechano-allodynia (FIG. 1). The development ofmechano-allodynia at 16 h was significantly attenuated by LT1002, butnot by LT1017 (FIG. 1).

Paclitaxel-Induced Neuropathic Pain and Drug Administration: The wellcharacterized rat model developed by Bennett was used, in which repeatedintraperitoneal (i.p) injections of low doses of paclitaxel induceneuropathic pain (mechano-allodynia) with little systemic toxicity ormotor impairment. The behavioral responses last for several weeks tomonths, thus modeling painful neuropathies in patients. Paclitaxel orits vehicle (Cremophor EL and 95% dehydrated ethanol in 1:1 ratio) wasinjected i.p in rats on four alternate days that is day (D) 0, 2, 4 and6 at 1 mg/kg on with a final cumulative dose of 4 mg/kg. The followingexperimental test substances were used: LT1002, LT1017, 504B3 andLT1015; these were dissolved in saline and provided by Lpath inindividual vials. Experimental test substances were given intravenously(i.v) at 25 mg/kg according to a dosing regimen designed by Lpath asfollows (and see experimental design, schematic in power point format).Experimental test substances were given one day before (D−1) the firstinjection of paclitaxel, and subsequently on D2, D5, D8, D11 and D14after the first injection of paclitaxel. Vehicle that was used todissolve test substance (saline) was injected according to the samedosing paradigm in paclitaxel-treated group or its respective vehicle.If injections of experimental test substances coincided with theinjection of paclitaxel (i.e. D2), experimental test substances weredelivered 15 min before paclitaxel. Mechanical withdrawal thresholdswere assessed with an electronic version of the von Frey test (dynamicplantar aesthesiometer, model 37450; Ugo Basile, Milan, Italy) on D−1before experimental test substance injection, the day after (D0) andbefore the first i.p. injection of paclitaxel and subsequently on D12and D16. To this end, each rat was placed in a Plexiglas chamber(28×40×35-cm, wire mesh floor) and allowed to acclimate for fifteenminutes. After acclimation, a servo-controlled mechanical stimulus (apointed metallic filament) was applied to the plantar surface, whichexerts a progressively increasing punctate pressure, reaching up to 50 gwithin 10 s. The pressure evoking a clear voluntary hind-paw withdrawalresponse was recorded automatically and taken as the mechanicalthreshold index. Mechanical threshold is assessed three times at eachtime point to yield a mean value, which is reported as mean absolutethreshold (grams, g). The development of mechano-allodynia is evidencedby a significant (P<0.05) reduction in mechanical mean absolutepaw-withdrawal thresholds (grams, g) at forces that failed to elicitwithdrawal responses before paclitaxel treatment (baseline). Paclitaxeltreatments results in bilateral allodynia.¹⁻³ Because thresholds did notdiffer between left and right hind paws at any time point in any group,values from both paws were averaged for further analysis and datapresentation. A total of four groups were used with n=3 rats/group.

-   Group 1: Vehicle instead of paclitaxel+saline-   Group 2: Paclitaxel+saline-   Group 3: Paclitaxel+LT1002-   Group 4: Paclitaxel+LT 1017-   Group 5: Paclitaxel+504B3-   Group 6: Paclitaxel+LT1015

Paw withdrawal threshold (g) for groups 1-6 on (D−1) and before i.vinjection of experimental test substances or their vehicle (saline) were(mean+/s.em): 43.6±0.296, 43.3±0.376, 43.7±0.333 and 42.9±0.219,respectively.

Statistical Analysis. Data are expressed as mean±SEM for 3 animals pergroup and analyzed by two-tailed, two-way ANOVA with Bonferroni post hoccomparisons to the paclitaxel group. *P<0.05, **P<0.001 paclitaxel vs.vehicle group.

Results: effects of LT1002 and LT1017 on paclitaxel-induced neuropathicpain. When compared to the vehicle treated group, administration ofpaclitaxel led to the development of mechano-allodynia (FIG. 1). Thedevelopment of mechano-allodynia at 16 h was significantly attenuated byLT1002, but not by the isotype control, LT1017 (FIG. 1). Thus anantibody to S1P was able to attenuate the development of neuropathicpain resulting from chemotherapeutic treatment.

Example 2 Antibody to S1P Reduces Thermal Hyperalgesia

As reviewed supra, intraplantar injection of ceramide in rats leads toperipheral sensitization and mechanical hyperalgesia (Joseph and Levine,2004); S1P contributes to excitation of rat sensory neurons and ceramidelevels can be at least partly regulated by conversion of ceramide tosphingosine and from there to S1P. For these reasons the role of S1P inceramide induced thermal hyperalgesia was studied, using the anti-S1Pantibody, LT1002. Doyle et al. (2011) J. Neurosci. 499:4-8.

Male Sprague Dawley rats (200-220 g) were purchased from Harlan(Indianapolis, Ind.), housed 3-4 per cage, and maintained in acontrolled environment (12 h light/dark cycles) with food and water adlibitum. The murine anti-S1P monoclonal antibody (LT1002) or itsisotype-matched control monoclonal antibody (LT1017) or their vehicle(saline) were given by intraplantar injection into the right hindpaw ofrats 15 minutes before intraplantar injections of C₂ ceramide(D-erythro-Sphingosine, N-Acetyl, Calbiochem, La Jolla Calif.) or itsvehicle, DMSO. Drugs were injected in a 5 ul injection volume using aHamilton guge needle (3.5″) in lightly anesthetized rats (80% CO₂/20%O₂). Hyperalgesic responses to heat were determined by the Hargreaves'Method using a Basile Plantar Test (Ugo Basile, Comeria, Italy) with acut-of latency of 20 s to prevent tissue damage. Hargreaves et al.(1988) Pain 32:77-88. Rats were individually confide to Plexiglaschambers and allowed to acclimate for 15 minutes prior to behavioraltesting. A mobile infrared generator was positioned to deliver a thermalstimulus directly to an individual hindpaw from beneath the chamber. Thewithdrawal latency period of injected paws was determined with anelectronic clock circuit and thermocouple. Two readings were taken foreach paw to calculate a mean latency for each animal. Thermalhyperalgesia results are the mean latency for each group and areexpressed as Paw Withdrawal Latency (PWL). All treatments were conductedwith the experimenters blinded to treatment conditions.

Intraplantar injection of ceramide (10 ug, n=4), given at a dosepreviously shown to elicit mechanical and thermal hyperalgesia [Doyle etal (2011) FASEB J 25: 2782-2791, Joseph et al (2004) J. Neurosci.20:2896-2902] led to a time-dependent development of thermalhyperalgesia that peaked by 2 hr. As shown in FIG. 2, This effect wasblocked by anti-S1P antibody LT1002 (242 ug, n=3) but not by theisotype-matched control monoclonal antibody LT1017. When tested alone,neither the anti-S1P antibody (242 ug) nor the control antibody (286 ug)had any effect on baseline withdrawal latencies. S1P contributes to thehyperalgesic responses to ceramide, and thus S1P inhibitors such asantibody inhibitors of S1P are believed to be therapeutically useful inpain relief, including relief of inflammatory pain.

All of the compositions and methods described and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods. All such similar substitutes and modificationsapparent to those skilled in the art are deemed to be within the spiritand scope of the invention as defined by the appended claims.

All patents, patent applications, and publications mentioned in thespecification are indicative of the levels of those of ordinary skill inthe art to which the invention pertains. All patents, patentapplications, and publications, including those to which priority oranother benefit is claimed, are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

What is claimed is:
 1. A method of treating or preventing pain in ahuman subject, comprising administering to a human subject having orbelieved to be at risk of having pain an antibody or fragment thereofthat binds and neutralizes S1P, thereby effecting treatment orprevention of pain.
 2. The method of claim 1 wherein the pain isneuropathic pain, allodynia, or hyperalgesia.
 3. The method of claim 1wherein the neuropathic pain is chemotherapeutic drug-inducedneuropathic pain.
 4. The method of claim 1 wherein the antibody is amonoclonal antibody.
 5. The method of claim 4 wherein the antibody is ahumanized antibody.